Protein formulations with reduced viscosity and uses thereof

ABSTRACT

Protein formulations and methods for reducing the viscosity of a protein formulation are provided. The method for reducing the viscosity of a protein formulation comprises adding a viscosity reducing agent, such as calcium chloride or magnesium chloride to the protein formulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional U.S. Application Ser.No.60/752660, filed on Dec. 21, 2005, which is herein incorporated byreference in its entirety.

TECHNICAL FIELD

The field relates to protein formulations and, more particularly, toprotein formulations with reduced viscosity.

BACKGROUND

It is estimated that more than 371 new biotechnology-based medicines arein the industry pipeline. Such biotechnology-based medicines includetherapeutic proteins such as enzymes, soluble receptors, ligands, bloodproteins, and monoclonal antibodies. Protein-based therapy, especiallymonoclonal antibody-based therapy, has become an important method fortreating diseases such as cancer, allergic diseases, asthma, and organtransplantation. At the end of 2003 fourteen antibody-based therapieshad been approved by the Food and Drug Administration to treat differenthuman diseases.

Antibody-based therapy is usually administered on a regular basis andrequires several mg/kg dosing by injection. Subcutaneous injection is atypical route of administration of these therapies. Because of the smallvolumes used for subcutaneous injection (usually 1.0 mL-1.2 mL), forhigh dose antibody therapies, this route of administration requires highconcentration protein formulations (e.g., 50 mg/ml-300 mg/ml).

High protein concentrations pose challenges relating to the physical andchemical stability of the protein, and difficulty with manufacture,storage, and delivery of the protein formulation. One problem is thetendency of proteins to form particulates during processing and/orstorage, which make manipulation during further processing difficult. Toattempt to obviate this problem, surfactants and/or sugars have beenadded to protein formulations. Although surfactants and sugars mayreduce the degree of particulate formation of proteins, they do notaddress another problem associated with manipulating and administeringconcentrated protein formulations, i.e., increased viscosity. In fact,sugars may enhance the intermolecular interactions within a protein orbetween proteins and increase the viscosity of the protein formulation.

Increased viscosity of protein formulations has negative ramificationsfrom processing through drug delivery to the patient. Accordingly, thereis a need in the art to develop relatively high concentration proteinformulations with suitably low viscosities that are suitable formanufacture, storage, and administration.

SUMMARY

The instant application relates to protein formulations having reducedviscosity compared to a corresponding protein formulation that does notinclude a viscosity-reducing agent in a suitable concentration, andmethods of making such protein formulations having reduced viscosity(reduced viscosity formulations).

In one aspect, the invention relates to methods of reducing theviscosity of a protein formulation by adding a viscosity reducing agentto a protein formulation, thereby reducing the viscosity of the proteinformulation compared to a protein formulation lacking the viscosityreducing agent. In one embodiment, the method involves determining theviscosity of a protein formulation prior to the addition of a viscosityreducing agent. In another embodiment, the method involves determiningthe viscosity of a protein formulation after the addition of a viscosityreducing agent. In yet another embodiment, the method involvesdetermining the viscosity of a protein formulation prior to and afterthe addition of a viscosity reducing agent. In certain embodiments, theviscosity reducing agent reduces the viscosity of the proteinformulation by at least 5% compared to the viscosity of the formulationformulated without the viscosity reducing agent.

In some embodiments, the viscosity reducing agent is calcium chloride ormagnesium chloride. The viscosity reducing agent is added at lowconcentrations so as not to negatively impact the protein formulation.The viscosity reducing agent is generally added to a protein formulationto a final concentration of between about 1 mM and about 50 mM. In someembodiments, the viscosity reducing agent is added to a proteinformulation to a final concentration of between about 5 mM and about 25mM. In certain embodiments, the viscosity reducing agent is added to aprotein formulation to a final concentration of between about 1 mM andabout 20 mM. In certain embodiments, the viscosity reducing agent isadded to a protein formulation to a final concentration of between 0.5mM and 14 mM. In another embodiment, the protein is an antibody, an Igfusion protein, a receptor, a ligand, a transcription factor, an enzyme,or a biologically active fragment thereof. In some embodiments, theprotein is an anti-myostatin antibody, an anti-IL-12 antibody, or ananti-IL-13 antibody.

In another aspect, the invention relates to a reduced viscosity proteinformulation. The reduced viscosity protein formulation includes aprotein, a viscosity reducing agent, and a buffer. In some embodiments,the viscosity reducing agent is calcium chloride or magnesium chloride.The viscosity reducing agent is generally added to a protein formulationto a final concentration of between about 1 mM and about 50 mM. In someembodiments, the viscosity reducing agent is added to a proteinformulation to a final concentration of between about 5 mM and about 25mM. In certain embodiments, the viscosity reducing agent is added to aprotein formulation to a final concentration of between about 1 mM andabout 15 mM. In certain other embodiments, the viscosity reducing agentis added to a protein formulation to a final concentration of between0.5 mM and 14 mM. When the viscosity reducing agent is added to aprotein formulation to a concentration of between about 0.5 mM to about50 mM, sodium chloride and sodium biphosphate are not used as viscosityreducing agents. The pH of the protein formulation is generally betweenabout 5.5 and about 6.5. In certain embodiments, the protein is anantibody, an Ig fusion protein, a receptor, a ligand, a transcriptionfactor, an enzyme, or a biologically active fragment thereof. In certainembodiments, the protein formulations are provided as kits. Such kitscan include instructions for use of the protein formulation.

In certain embodiments, the reduced viscosity protein formulation is areduced viscosity anti-myostatin antibody formulation. In oneembodiment, the anti-myostatin antibody is a monoclonal antibody. Inanother embodiment, the anti-myostatin antibody is a humanizedmonoclonal antibody (e.g., a partially humanized or fully humanizedmonoclonal antibody). In certain embodiments, the anti-myostatinantibody is MYO-022, MYO-028 or MYO-029. Anti-myostatin antibodies aregenerally used at a concentration of between about 25 mg/ml to about 400mg/ml. The viscosity reducing agent is generally added to a reducedviscosity anti-myostatin antibody formulation to a final concentrationof between about 1 mM and about 50 mM. In some embodiments, theviscosity reducing agent is added to an anti-myostatin antibody to afinal concentration of between about 5 mM and about 25 mM. In certainembodiments, the viscosity reducing agent is added to an anti-myostatinantibody formulation to a final concentration of between about 1 mM andabout 15 mM. In certain embodiments, the viscosity reducing agent isadded to an anti-myostatin antibody formulation to a final concentrationof between 0.5 mM and 14 mM. When the viscosity reducing agent is addedto an anti-myostatin antibody formulation to a concentration of betweenabout 0.5 mM to about 50 mM, sodium chloride and sodium biphosphate arenot used as viscosity reducing agents. Reduced viscosity anti-myostatinantibody formulations generally have a pH of between about 5.5 and about6.5. In one embodiment, histidine is used to buffer a reduced viscositymyostatin antibody formulation. A reduced viscosity myostatin antibodyformulation can also include one or more cryoprotectants, one or moresurfactants, one or more anti-oxidants, or a combination thereof. Insome embodiments, the reduced viscosity anti-myostatin formulation is areconstituted formulation. Myostatin antibodies can be formulated asdescribed herein as pharmaceutical compositions and used to treatdisorders such as, but not limited to, muscular dystrophy, sarcopenia,cachexia, and Type II diabetes. In certain embodiments, a reducedviscosity anti-myostatin antibody formulation is provided as a kit. Suchkits can include instructions for use of the antibody formulation.

In certain embodiments, the reduced viscosity protein formulation is areduced viscosity anti-IL-12 antibody formulation. In one embodiment,the anti-IL-12 antibody is a monoclonal antibody. In another embodiment,the anti-IL-12 antibody is a humanized monoclonal antibody (e.g., apartially humanized or fully humanized monoclonal antibody). In certainembodiments, the anti-IL-12 antibody is J695. Anti-IL-12 antibodies aregenerally used in a formulation at a concentration of between about 25mg/ml to about 400 mg/ml. A viscosity reducing agent is generally addedto an anti-IL-12 antibody formulation to a final concentration ofbetween about 1 mM and about 50 mM. In some embodiments, the viscosityreducing agent is added to an anti-IL-12 antibody formulation to a finalconcentration of between about 5 mM and about 25 mM. In certainembodiments, the viscosity reducing agent is added to an anti-IL-12antibody formulation to a final concentration of between about 1 mM andabout 15 mM. In certain other embodiments, the viscosity reducing agentis added to an anti-IL-12 antibody formulation to a final concentrationof between 0.5 mM and about 14 mM. When the viscosity reducing agent isadded to an anti-IL-12 antibody formulation to a concentration ofbetween about 0.5 mM to about 50 mM, sodium chloride and sodiumbiphosphate are not used as viscosity reducing agents. Reduced viscosityanti-IL-12 antibody formulations generally have a pH of between about5.5 and about 6.5. In certain embodiments, histidine is used as a bufferin a reduced viscosity IL-12 antibody formulation. Reduced viscosityanti-IL-12 antibody formulations can also include one or morecryoprotectants, one or more surfactants, one or more anti-oxidants, orcombinations thereof. In some embodiments, the reduced viscosityanti-IL-12 formulation is a reconstituted formulation. Anti-IL-12antibodies can be formulated as described herein for use aspharmaceutical compositions and used to treat disorders such as, but notlimited to, rheumatoid arthritis, Crohn's disease, psoriasis, andpsoriatic arthritis. In certain embodiments, a reduced viscosityanti-IL-12 antibody formulation is provided as part of a kit. Such kitscan include instructions for use of the anti-IL-12 antibody formulation.

In certain embodiments, the reduced viscosity protein formulation is ananti-IL-13 antibody formulation. In one embodiment, the anti-IL-13antibody is a monoclonal antibody. In another embodiment, the anti-IL-13antibody is a humanized monoclonal antibody (e.g., partially humanizedor fully humanized). In certain embodiments, the anti-IL-13 antibody isIMA-638. Anti-IL-13 antibodies are generally used in a formulation at aconcentration of between about 25 mg/ml to about 400 mg/ml. A viscosityreducing agent is generally added to make a reduced viscosity anti-IL-13antibody formulation to a final concentration of between about 1 mM andabout 50 mM. In some embodiments, the viscosity reducing agent is addedto an anti-IL-13 antibody formulation to a final concentration ofbetween about 5 mM and about 25 mM. In certain embodiments, theviscosity reducing agent is added to an anti-IL-13 antibody formulationto a final concentration of between about 1 mM and about 15 mM. Incertain other embodiments, the viscosity reducing agent is added to ananti-IL-13 antibody formulation to a final concentration of between 0.5mM and about 14 mM. When the viscosity reducing agent is added to ananti-IL-13 antibody formulation to a concentration of between about 0.5mM to about 50 mM, sodium chloride and sodium biphosphate are not usedas viscosity reducing agents. Reduced viscosity anti-IL-13 antibodyformulations generally have a pH of between about 5.5 and about 6.5. Inone embodiment, histidine is used as a buffer in a reduced viscosityIL-13 antibody formulation. A reduced viscosity anti-IL-13 antibodyformulation can also include one or more cryoprotectants, one or moresurfactants, one or more anti-oxidants, or combinations thereof. In someembodiments, the reduced viscosity anti-IL-13 formulation is areconstituted formulation. Anti-IL-13 antibodies can be formulated in areduced viscosity formulation as pharmaceutical composition and used totreat disorders such as, but not limited to, respiratory disorders(e.g., asthma); atopic disorders (e.g., allergic rhinitis); inflammatoryand/or autoimmune conditions of the skin (e.g., atopic dermatitis),gastrointestinal organs (e.g., inflammatory bowel diseases (IBD)), aswell as fibrotic and cancerous disorders. In certain embodiments, areduced viscosity anti-IL-13 antibody formulation is provided as a kit.Such kits can include instructions for use of the reduced viscosityanti-IL-13 antibody formulation.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the results of experiments conducted todetermine the effect of increasing concentrations of various salts onthe viscosity of an anti-myostatin (MYO-029) antibody formulation.

FIG. 2 is a bar graph depicting the results of experiments conducted todetermine the effect of increasing concentrations of calcium chloride onthe viscosity of an anti-myostatin (MYO-028) antibody formulation.

FIG. 3 is a graph depicting the results of experiments conducted todetermine the effect of increasing concentrations of calcium chloride onthe viscosity of an anti-IL-13 (IMA-638) antibody formulation.

FIG. 4A is a bar graph depicting the results of experiments conducted totest the effect of freeze-thaw-induced degradation of the anti-myostatin(MYO-029) antibody in the presence or absence of calcium chloride.Degradation was assessed by protein recovery as determined by measuringabsorbance at 280 nm.

FIG. 4B is a bar graph depicting the results of experiments conducted totest the effect of freeze-thaw-induced degradation of the anti-myostatin(MYO-029) antibody in the presence or absence of calcium chloride.Degradation was assessed by percentage of high molecular weight species(% HMW) formation as determined by size exclusion-high performanceliquid chromatography (SEC-HPLC).

FIG. 5A is a bar graph depicting the results of experiments conducted totest the effect of the presence or absence (control) of calcium chlorideon the liquid stability of anti-myostatin (MYO-029) antibody subjectedto storage at 40° C. for up to seven days. Liquid stability of MYO-029antibody was determined by measuring absorbance at 280 nm.

FIG. 5B is a bar graph depicting the results of experiments conducted totest the effect of the presence or absence (control) of calcium chlorideon the liquid stability of anti-myostatin (MYO-029) antibody subjectedto storage at 40° C. for up to seven days. Liquid stability of MYO-029antibody was determined by measuring HMW formation as determined bySEC-HPLC.

FIG. 6 is a representation of the amino acid sequence of the MYO-028antibody heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2).

FIG. 7 is a representation of the amino acid sequence of the MYO-029antibody heavy chain (SEQ ID NO:3) and light chain (SEQ ID NO:4).

FIG. 8 is a representation of the amino acid sequence of the J695antibody heavy chain (SEQ ID NO:5) and light chain (SEQ ID NO:6).

FIG. 9 is a representation of the amino acid sequence of the IMA-638antibody heavy chain (SEQ ID NO:7) and light chain (SEQ ID NO:8). Thelast amino acid residue encoded by the heavy chain DNA sequence, Lys⁴⁴⁸,is observed in the mature, secreted form of IMA-638 only in smallquantities and is presumably removed from the bulk of the monoclonalantibody during intracellular processing by CHO cellular proteases.Therefore, the carboxy-terminus of the IMA-638 heavy chain is Gly⁴⁴⁷.Carboxy-terminus lysine processing has been observed in recombinant andplasma-derived antibodies and does not appear to impact their function.

DETAILED DESCRIPTION

The viscosity of a protein formulation has implications for thestability, processing, storage, and, for those used as drugs, drugdelivery of the protein formulation to a patient. Such implicationsinclude, but are not limited to concentration and buffer exchange viaultrafiltration and diafiltration (the flux across the membrane maydecrease with increasing viscosity thereby resulting in longerprocessing times), sterile filtration (it takes longer to sterile filterviscous solutions, and in some instances a very viscous solution willnot pass through membranes with very small pores, e.g., 0.22 μmmembranes), sample handling (e.g., difficulty with pipetting and theability to draw into a syringe), recovery from the storage vial postreconstitution, stability, and passage through needles for subcutaneousor intramuscular administration.

Provided herein are methods of reducing the viscosity of a proteinformulation that have been identified. The methods are suitable forpreparing protein formulations having reduced viscosity (“reducedviscosity formulations” or “reduced viscosity protein formulations”).These reduced viscosity protein formulations include a protein ofinterest and a viscosity reducing agent.

Methods of Reducing the Viscosity of a Protein Formulation

The term “viscosity” as used herein, may be “kinematic viscosity” or“absolute viscosity.” “Kinematic viscosity” is a measure of theresistive flow of a fluid under the influence of gravity. When twofluids of equal volume are placed in identical capillary viscometers andallowed to flow by gravity, a viscous fluid takes longer than a lessviscous fluid to flow through the capillary. If one fluid takes 100seconds to complete its flow and another fluid takes 200 seconds, thesecond fluid is twice as viscous as the first on a kinematic viscosityscale. “Absolute viscosity,” sometimes called “dynamic” or “simpleviscosity,” is the product of kinematic viscosity and fluid density. Thedimension of kinematic viscosity is L²/T where L is a length and T is atime. Commonly, kinematic viscosity is expressed in centistokes (cSt).The SI unit of kinematic viscosity is mm²/s, which is 1 cSt. Absoluteviscosity is expressed in units of centipoise (cP). The SI unit ofabsolute viscosity is the millipascal-second (mPa·s), where 1 cP=1mPa·s.

The viscosity of a protein formulation can be reduced by the addition ofa viscosity reducing agent to the formulation. In some cases, theviscosity reducing agent is added at a relatively low concentration. Theviscosity of a formulation comprising a viscosity reducing agent isreduced compared to the viscosity of a formulation lacking the viscosityreducing agent. When the addition of the viscosity reducing agentresults in lowering the viscosity of the formulation compared to acorresponding formulation that does not include the viscosity reducingagent or compared to a formulation that does not include the viscosityreducing agent at a selected concentration, the formulation containingthe viscosity reducing agent (e.g., in a selected concentration), theformulation is a reduced viscosity formulation. In certain reducedviscosity formulations, the viscosity reducing agent generally reducesthe viscosity of a protein formulation by about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, and about 90% compared to the viscosity of a proteinformulation without, or containing lower amounts of, the viscosityreducing agent. In some cases, the viscosity reducing agent reduces theviscosity of a protein formulation by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, andat least 90% compared to the viscosity of a protein formulation without,or containing lower amounts of, the viscosity reducing agent. In certainembodiments, the viscosity of a protein formulation is measured prior tothe addition of the viscosity reducing agent. In other embodiments, theviscosity of a protein formulation is measured after the addition of theviscosity reducing agent. Such measurements may be made hours (e.g.,1-23 hours), days (e.g., 1-10 days), weeks (e.g., 1-5 weeks), or months(e.g., 1-12 months), or years (e.g., 1-2 years, 1-3 years) after theaddition of a viscosity reducing agent to a protein formulation. In yetother embodiments, the viscosity of the protein formulation is measuredprior to and after the addition of the viscosity reducing agent. Methodsof measuring viscosity are well known in the art and include, forexample, using a capillary viscometer, or a cone-plate rheometer.

In one embodiment, the viscosity reducing agent is a salt such ascalcium chloride, magnesium chloride, sodium phosphate, or argininehydrochloride. In the method described herein, the viscosity reducingagent is added to the protein formulation to a final concentration ofbetween about 0.5 mM and about 100 mM. In one embodiment, the viscosityreducing agent is added to the protein formulation to a finalconcentration of between about 5 mM and about 20 mM. In anotherembodiment, the viscosity reducing agent is added to the proteinformulation to a final concentration of between 0.5 mM and 14 mM. Incertain embodiments, the viscosity reducing agent is added to theprotein formulation to a final concentration of between about 0.5 mM andnot greater than 20 mM, or 19 mM, or 18 mM, or 17 mM, or 16 mM, or 15mM, or 14 mM, or 13 mM, or 12 mM, or 11 mM, or 10 mM. In general, whenthe viscosity reducing agent is added to the protein formulation to afinal concentration of between about 0.5 mM and about 25 mM, theviscosity reducing agent is calcium chloride or magnesium chloride, butnot sodium chloride, or sodium biphosphate. In certain embodiments, theviscosity reducing agent is added at low concentrations so as not tonegatively impact the protein formulation. For example, at calciumchloride or magnesium chloride concentrations of 20 mM or greater,proteins may form a gel at low storage temperatures (e.g., 2-8° C.).Accordingly, a concentration of a viscosity reducing agent is generallyselected for which the viscosity is reduced at the intended storagetemperature of the reduced viscosity formulation.

Formulations

The composition of a reduced viscosity protein formulation is determinedby consideration of several factors. These factors include, but are notlimited to: the nature of the protein (e.g., receptor, antibody, Igfusion proteins, enzyme); the concentration of the protein; the desiredpH range; how the protein formulation is to be stored (e.g.,temperature); the period of time over which the protein formulation isto be stored; and how the formulation is to be administered to apatient. The selection of an appropriate viscosity reducing agent ismade based, in part, on such requirements for the protein in theformulation.

Proteins

The protein of interest to be formulated includes, but is not limitedto, proteins such as, myostatin/GDF-8; interleukins (ILs), e.g., IL-1 toIL-15; growth hormones such as human growth hormone and bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; uricase; bikunin; bilirubin oxidase; subtilisin;lipoproteins; α-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; Factor Vlla; Factor VIII, Factor VIIIC; Factor IX;tissue factor; von Willebrand factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or tissue-type plasminogen activator(t-PA); bombazine; thrombin; plasmin; miniplasmin; microplasmin; tumornecrosis factor-α and -β; enkephalinase; RANTES (Regulated on ActivationNormally T-cell Expressed and Secreted); human macrophage inflammatoryprotein (MIP-1-α); serum albumin such as human serum albumin;Mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin;activin; vascular endothelial growth factor (VEGF); placental growthfactor (PIGF); receptors for hormones or growth factors; an integrin;protein A or protein D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-α and TGF-β, including TGF-β 1, TGF-β 2, TGF-β 3,TGF-β 4, or TGF-β 5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factorbinding proteins; CD proteins such as: CD2, CD3, CD4, CD8, CD9, CD19,CD20, CD22, CD28, CD34, and CD45; erythropoietin (EPO); thrombopoietin(TPO); osteoinductive factors; immunotoxins; a bone morphogeneticprotein (BMP); an interferon such as interferon-α, -β, and -γ; a colonystimulating factor (CSF), e.g., M-CSF, GM-CSF, and G-CSF; superoxidedismutase; T-cell receptors; members of the HER receptor family such asthe EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion moleculessuch as LFA-1, VLA-4, ICAM-1, and VCAM; IgE; blood group antigens;flk2/flt3 receptor; obesity (OB) receptor; decay accelerating factor(DAF); a viral antigen such as, HIV gag, env, pol, tat, or rev proteins;homing receptors; addressins; immunoadhesins; and biologically activefragments, or variants of any of the above-listed polypeptides. In someformulations, more than one type of protein or fragment is included inthe formulation.

The term “biologically active fragment” means a fragment of a proteinthat retains at least one of the functions of the protein from which itis derived. A biologically active fragment of an antibody includes anantigen-binding fragment of the antibody; a biologically active fragmentof a receptor includes a fragment of the receptor that can still bindits ligand; a biologically active fragment of a ligand includes thatportion of a ligand that can still bind its receptor; and a biologicallyactive fragment of an enzyme includes that portion of the enzyme thatcan still catalyze a reaction catalyzed by the full length enzyme. Inone embodiment, a biologically active fragment retains at least about 5%of the function of the protein from which it is derived. The function ofa protein can be assayed by methods known in the art (e.g., testingantibody-antigen interactions, testing ligand-receptor interactions,testing enzymatic activity, testing transcriptional activity, or testingDNA-protein interactions). In some cases, the fragment is atherapeutically useful fragment, which may, for example, retain certainfeatures of the protein from which it is derived (e.g., binding to aspecific ligand) but does not cause cellular response elicited by theprotein from which it is derived.

In certain embodiments, the protein to be formulated is an antibody. Theantibody may be one that can bind to one of the above-mentionedproteins. The term “antibody” as used herein, includes polyclonalantibodies, monoclonal antibodies, antibody compositions withpolyepitope specificities, bispecific antibodies, diabodies, or otherpurified preparations of antibodies and recombinant antibodies. Theantibodies can be whole antibodies, e.g., of any isotype (IgG, IgA, IgE,IgM, etc.), or fragments thereof, which bind the antigen of interest. Ina specific example of an antibody used in the present invention, theantibody to be formulated is an antibody having the IgG isotype.Antibodies can be fragmented using conventional or other techniques andthe fragments screened for binding to an antigen of interest. Generally,an antibody fragment comprises the antigen-binding and/or the variableregion of an intact antibody. Thus, the term antibody fragment includessegments of proteolytically cleaved or recombinantly prepared portionsof an antibody molecule that are can selectively bind to a selectedprotein. Non-limiting examples of such proteolytic and/or recombinantfragments include Fab, F(ab′)₂, Fab′, Fv, and single chain antibodies(scFv) containing a V[L] and/or V[H] domain joined by a peptide linker.The scFvs may be covalently or noncovalently linked to form antibodieshaving two or more binding sites.

In some embodiments, the antibody is a humanized monoclonal antibody.The term “humanized monoclonal antibody” as used herein, is a monoclonalantibody from a non-human source (recipient) that has been altered tocontain at least one or more of the amino acid residues found in theequivalent human monoclonal antibody (donor). A “fully humanizedmonoclonal antibody” is a monoclonal antibody from a non-human sourcethat has been altered to contain all of the amino acid residues found inthe antigen-binding region of the equivalent human monoclonal antibody.Humanized antibodies may also comprise residues that are not foundeither in the recipient antibody or the donor antibody. Thesemodifications can be made to further refine and optimize antibodyfunctionality. A humanized antibody may also optionally comprise atleast a portion of a human immunoglobulin constant region (Fc).

In certain embodiments, an antibody used in a reduced viscosityformulation is an anti-myostatin antibody (e.g., MYO-022, MYO-028 (FIG.6), MYO-029 (FIG. 7)). MYO-022, MYO-028, and MYO-029 antibodies aredescribed in U.S. patent application Ser. No. 10/688,925 (Pub. No.2004/0142382), which is incorporated herein by reference. In otherembodiments, the antibody is an IL-12 antibody (e.g., J695 (FIG. 8)).The J695 antibody is described in U.S. Pat. No. 6,914,128, which isincorporated herein by reference. In yet another embodiment, theantibody is an anti-IL-13 antibody (e.g., IMA-638 (FIG. 9), CAT-354).Anti-IL-13 antibodies are described in U.S. patent application Ser. No.11/149,309, which is incorporated herein by reference.

In some embodiments, the protein to be formulated is a fusion protein.In one embodiment, the fusion protein is an immunoglobulin (Ig) fusionprotein. In a specific embodiment, the fusion protein comprises the IgGheavy chain constant region. In another embodiment, the fusion proteincomprises an amino acid sequence corresponding to the hinge, CH2 and CH3regions of human immunoglobulin Cγl. Examples of Ig fusion proteinsinclude CTLA4 Ig and VCAM2D-IgG. Methods of making fusion proteins areknown in the art (e.g., U.S. Pat. Nos. 6,887,471 and 6,482,409).

In certain embodiments, the protein to be formulated is a protein thatdoes not include a Factor VII polypeptide, or anti-IgE antibody.

A reduced viscosity formulation can contain more than one protein asnecessary for the treatment of a particular disorder. The additionalprotein(s) typically have complementary activities to the otherprotein(s) in the formulation, and do not adversely affect the otherprotein(s) in the formulation. For example, it may be desirable toprovide a single formulation containing two or more antibodies that bindto myostatin; two or more antibodies that bind to IL-12; or two or moreantibodies that bind to IL-13. In addition, a protein formulation canalso contain non-protein substances that are of use in the ultimateutility of the reduced viscosity protein formulation. For example,sucrose can be added to enhance stability and solubility of the proteinin solution; and histidine can be added to provide appropriate buffercapacity. Such additional substances can be part of a proteinformulation prior to addition of a viscosity reducing agent or added inthe process for making a reduced viscosity formulation.

In certain embodiments, the protein to be formulated is essentially pureand/or essentially homogeneous (i.e., substantially free fromcontaminating proteins, etc.) prior to its use in the formulation. Theterm “essentially pure” protein means a composition comprising at leastabout 90% by weight of a selected protein fraction, for example at leastabout 95% by weight of the selected protein fraction. The term“essentially homogeneous” protein means a composition comprising atleast about 99% by weight of a selected protein fraction, excluding themass of various stabilizers and water in solution.

Concentration of the Protein in a Low Viscosity Formulation

The concentration of the protein in a reduced viscosity formulation isdependent on the ultimate use of the formulation. Protein concentrationsin the formulations described herein are generally between about 10mg/ml and about 300 mg/ml, e.g., between about 10 mg/ml and about 100mg/ml, about 25 mg/ml and about 100 mg/ml, about 50 mg/ml and about 100mg/ml, about 75 mg/ml and about 100 mg/ml, about 100 mg/ml and about 200mg/ml, about 125 mg/ml and about 200mg/ml, about 150 mg/ml and about 200mg/ml, about 200 mg/ml and about 300 mg/ml, and about 250 mg/ml andabout 300 mg/ml. For example, protein concentrations in the formulationsdescribed herein can be between 10 mg/ml and 300 mg/ml, e.g., between 10mg/ml and 100 mg/ml, between 25 mg/ml and 100 mg/ml, between 50 mg/mland 100 mg/ml, between 75 mg/ml and 100 mg/ml, between 100 mg/ml and 200mg/ml, between 125 mg/ml and 200mg/ml, between 150 mg/ml and 200 mg/ml,between 200 mg/ml and 300 mg/ml, and between 250 mg/ml and 300 mg/ml.The term “between” is intended to be inclusive of the minimal andmaximal concentrations.

Reduced viscosity protein formulations can be used for therapeuticpurposes. Accordingly, the concentration of the protein in a formulationused for a therapeutic application is determined based on providing theprotein in a dosage and volume that is tolerated by, and of therapeuticvalue to, the patient. If a reduced viscosity formulation is to beadministered by injection, the protein concentration will be dependenton the injection volume (usually 1.0 mL-1.2 mL). Protein based therapiescan require several mg/kg of dosing per week, per month, or per severalmonths. Accordingly, if a protein is to be provided at 2-3 mg/kg of bodyweight of the patient, and an average patient weighs 75 kg, 150 mg-225mg of the protein will need to be delivered in a 1.0 mL-1.2 mL injectionvolume. Alternatively, the formulation is provided in a concentrationsuitable for delivery at more than one injection site per treatment.

As the concentration of the protein in a formulation increases, theviscosity of the protein formulation is also likely to increase.Increased viscosity of the formulation makes the formulation harder toadminister. Accordingly, there is a need to decrease the viscosity ofprotein formulations when the increased viscosity impacts its ability tobe utilized.

Viscosity Reducing Agents

It has been found that adding relatively low concentrations of certainviscosity reducing agents to a protein formulation reduces the viscosityof the protein formulation. The term “viscosity reducing agent” as usedherein, includes any agent that reduces the viscosity of a proteinformulation compared to a protein formulation not containing, orcontaining a lesser amount of, the viscosity reducing agent. Forexample, a viscosity reducing agent generally reduces the viscosity of aprotein formulation by about 5%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 90%, or about 95% comparedto the viscosity of the protein formulation without, or containing loweramounts of, a viscosity reducing agent. For example, a viscosityreducing agent generally reduces the viscosity of the proteinformulation by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, or 95%compared to the viscosity of a protein formulation without, orcontaining lower amounts of, the viscosity reducing agent. Non-limitingexamples of viscosity reducing agents include calcium chloride,magnesium chloride, arginine hydrochloride, sodium chloride, sodiumthiocyanate, ammonium thiocyanate, ammonium sulphate, sodium phosphate,and ammonium chloride.

In one embodiment, the viscosity reducing agent is calcium chloride. Inanother embodiment, the viscosity reducing agent is magnesium chloride.In an alternate embodiment, more than one viscosity reducing agent isadded to a protein formulation.

A viscosity reducing agent is generally added to a protein formulationto a final concentration of between 1 mM to about 150 mM, e.g., betweenabout 1 mM and about 50 mM, between about 2 mM and about 40 mM, betweenabout 3 mM and about 30 mM, between about 4 mM and about 25 mM, betweenabout 5 mM and about 20 mM, between about 5 mM and about 25 mM, betweenabout 5 mM and about 30 mM, between about 5 mM and about 40 mM, andbetween about 5 mM and about 50 mM. In certain embodiments, theviscosity reducing agent is added to the protein formulation to a finalconcentration of less than 14 mM, less than 13 mM, less than 12 mM, lessthan 11 mM, less than 10 mM, less than 9 mM, less than 8 mM, less than 7mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, orless than 2 mM. In other embodiments, the viscosity reducing agent isadded to the protein formulation to a final concentration of between 0.5mM and 14 mM, between 0.5 mM and 13 mM, between 0.5 mM and 12 mM,between 0.5 mM and 11 mM, between 0.5 mM and 10 mM, between 0.5 mM and 9mM, between 0.5 mM and 8 mM, between 0.5 mM and 7 mM, between 0.5 mM and6 mM, or between 0.5 mM and 5 mM. In one embodiment, the viscosityreducing agent is calcium chloride at a final concentration of betweenabout 5 mM and about 20 mM in the formulation. In another embodiment,the viscosity reducing agent is calcium chloride at a finalconcentration of between 5 mM and about 14 mM in the formulation. Inother embodiments, the viscosity reducing agent is magnesium chloride ata final concentration of between about 5mM and about 20 mM in theformulation. In another embodiment, the viscosity reducing agent ismagnesium chloride at a final concentration of between 5 mM and about 14mM in the formulation.

The viscosity of a protein formulation can be measured by any suitablemethod known in the art including, for example, using a capillaryviscometer or a cone-plate rheometer.

Buffers

The term “buffer” as used herein, includes those agents that maintainthe pH of a solution, e.g., a formulation, in a desired range. The pH ofa formulation as described herein is generally between about pH 5.0 toabout 9.0, for example, about pH 5.5 to about 6.5, about pH 5.5 to about6.0, about pH 6.0 to about 6.5, pH 5.5, pH 6.0, or pH 6.5. In general, abuffer that can maintain a solution at pH 5.5 to 6.5 is used.Non-limiting examples of buffers that can be used in a formulationdescribed herein include, histidine, succinate, gluconate, tris(trometamol), phosphate, citrate, 2-morpholinoethanesulfonic acid (MES),sodium phosphate, sodium acetate, and cacodylate.

Histidine is a buffer that is typically in reduced viscosityformulations that are to be administered by subcutaneous, intramuscular,or peritoneal injection. The concentration of the buffer is betweenabout 5 mM and 30 mM. In one embodiment, the buffer of a formulation ishistidine at a concentration of about 5 mM to about 20 mM.

Excipients

In addition to the protein, a viscosity reducing agent, and buffer, areduced viscosity formulation as described herein may also contain othersubstances. Such substances include, but are not limited to,cryoprotectants, lyoprotectants, surfactants, bulking agents,anti-oxidants, and stabilizing agents. In one embodiment, a reducedviscosity protein formulation described herein includes an excipientselected from the group consisting of a cryoprotectant, a lyoprotectant,a surfactant, a bulking agent, an anti-oxidant, a stabilizing agent, andcombinations thereof.

The term “cryoprotectant” as used herein, includes agents that providestability to the protein in a formulation against freezing-inducedstresses, e.g., by being preferentially excluded from the proteinsurface. Cryoprotectants may also offer protection during primary andsecondary drying and long-term product storage. Non-limiting examples ofcryoprotectants include sugars, such as sucrose, glucose, trehalose,mannitol, mannose, and lactose; polymers, such as dextran, hydroxyethylstarch and polyethylene glycol; surfactants, such as polysorbates (e.g.,PS-20 or PS-80); and amino acids, such as glycine, arginine, leucine,and serine. A cryoprotectant exhibiting low toxicity in biologicalsystems is generally used. The cryoprotectant, if included in theformulation, is generally added to a final concentration of betweenabout 0.1% and about 10% (weight/volume), e.g., between about 0.5% andabout 10%, between about 0.5% and about 5%, between about 0.5% and about2%, between about 1% and about 5%, or between about 5% and about 10%. Inone embodiment, the cryoprotectant is sucrose at a concentration ofbetween about 0.5% and about 10% (weight/volume).

In one embodiment, a lyoprotectant is added to a formulation describedherein. The term “lyoprotectant” as used herein, includes agents thatprovide stability to the protein during the freeze-drying or dehydrationprocess (primary and secondary freeze-drying cycles), e.g., by providingan amorphous glassy matrix and by binding with the protein throughhydrogen bonding, replacing the water molecules that are removed duringthe drying process. This helps to maintain the protein conformation,minimize protein degradation during the lyophilization cycle, andimprove the long-term product stability. Non-limiting examples oflyoprotectants include sugars, such as sucrose or trehalose; an aminoacid, such as monosodium glutamate, non-crystalline glycine orhistidine; a methylamine such, as betaine; a lyotropic salt, such asmagnesium sulfate; a polyol, such as trihydric or higher sugar alcohols,e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, andmannitol; propylene glycol; polyethylene glycol; pluronics; andcombinations thereof. The amount of lyoprotectant added to a formulationis generally an amount that does not lead to an unacceptable amount ofdegradation/aggregation of the protein when the protein formulation islyophilized. Where the lyoprotectant is a sugar (such as sucrose ortrehalose) and the protein is an antibody, non-limiting examples oflyoprotectant concentrations in a reduced viscosity protein formulationare from about 10 mM to about 400 mM, from about 30 mM to about 300 mM,and from about 50 mM to about 100 mM.

In certain embodiments, a surfactant is included in a formulationdescribed herein. The term “surfactant” as used herein, includes agentsthat reduce the surface tension of a liquid by adsorption at theair-liquid interface. Examples of surfactants include, withoutlimitation, nonionic surfactants, such as polysorbates (e.g.,polysorbate 80 or polysorbate 20); poloxamers (e.g., poloxamer 188);Triton™ (e.g.,Triton™X-100); sodium dodecyl sulfate (SDS); sodium octylglycoside; lauryl-sulfobetaine; myristyl-sulfobetaine;linoleyl-sulfobetaine; stearyl-sulfobetaine; lauryl-sarcosine;myristyl-sarcosine; linoleyl-sarcosine; stearyl-sarcosine;linoleyl-betaine; myristyl- betaine; cetyl-betaine;lauroamidopropyl-betaine; cocamidopropyl-betaine;linoleamidopropyl-betaine; myristamidopropyl-betaine,palmidopropyl-betaine; isostearamidopropyl-betaine (e.g.,lauroamidopropyl); myristarnidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the Monaquat™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol; polypropyl glycol; and copolymers ofethylene and propylene glycol (e.g., pluronics, PF68). The amount ofsurfactant added is such that it maintains aggregation of thereconstituted protein at an acceptable level as assayed using, e.g.,SEC-HPLC to determine the percentage of high molecular weight (HMW)species or low molecular weight (LMW) species, and minimizes theformation of particulates after reconstitution of a lyophilate of aprotein formulation described herein. For example, the surfactant can bepresent in a formulation (liquid or prior to lyophilization) in anamount from about 0.001-0.5%, e.g., from about 0.05-0.3%.

In some embodiments, a bulking agent is included in a reduced viscosityformulation. The term “bulking agent” as used herein, includes agentsthat provide the structure of the freeze-dried product withoutinteracting directly with the pharmaceutical product. In addition toproviding a pharmaceutically elegant cake, bulking agents may alsoimpart useful qualities in regard to modifying the collapse temperature,providing freeze-thaw protection, and enhancing the protein stabilityover long-term storage. Non-limiting examples of bulking agents includemannitol, glycine, lactose, and sucrose. Bulking agents may becrystalline (such as glycine, mannitol, or sodium chloride) or amorphous(such as dextran or hydroxyethyl starch) and are generally used inprotein formulations in an amount from 0.5% to 10%.

Other pharmaceutically acceptable carriers, excipients, or stabilizers,such as those described in Remington: The Science and Practice ofPharmacy 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins(2000) may also be included in a protein formulation described herein,provided that they do not adversely affect the desired characteristicsof the formulation. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients (e.g., patients) at the dosages andconcentrations employed and include: additional buffering agents;preservatives; co-solvents; antioxidants, including ascorbic acid andmethionine; chelating agents such as EDTA; metal complexes (e.g.,Zn-protein complexes); biodegradable polymers, such as polyesters;salt-forming counterions, such as sodium, polyhydric sugar alcohols;amino acids, such as alanine, glycine, glutamine, asparagine, histidine,arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid,and threonine; organic sugars or sugar alcohols, such as lactitol,stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose,myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g.,inositol), polyethylene glycol; sulfur containing reducing agents, suchas urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol,α-monothioglycerol, and sodium thio sulfate; low molecular weightproteins, such as human serum albumin, bovine serum albumin, gelatin, orother immunoglobulins; and hydrophilic polymers, such aspolyvinylpyrrolidone.

Exemplary Protein Formulations

MYO-029

In one example, a MYO-029 reduced viscosity formulation can beformulated using 1 mg/ml to 300 mg/ml of the MYO-029 antibody. TheMYO-029 formulation generally includes between about 1 mM and about 50mM calcium chloride or magnesium chloride. The formulation can includeabout 5 mM to about 25 mM histidine. The formulation can include about1% to about 5% (w/v) sucrose or trehalose. In some instances, theformulation can include about 10 mM to about 25 mM methionine. Incertain MYO-029 formulations, 0.05-0.2% (w/v) polysorbate-20 orpolysorbate-80 is added. The pH of the formulation is generally between5.5 and 6.5. In a specific example, the MYO-029 formulation comprises150 mg/ml of the MYO-029 antibody, 10 mM calcium chloride or magnesiumchloride, 20 mM histidine, 4% sucrose, and has a pH of 6.0. In anotherspecific example, the MYO-029 formulation comprises 75 mg/ml of theMYO-029 antibody, 5 mM calcium chloride or magnesium chloride, 10 mMhistidine, 10 mM methionine, 2% sucrose, and has a pH of 6.0. In anotherspecific example, a MYO-029 antibody formulation comprises 150 mg/ml ofthe MYO-029 antibody, 10 mM calcium chloride or magnesium chloride, 20mM histidine, 20 mM methionine, 4% sucrose, 0.2% polysorbate-80, and hasa pH of 6.0.

MYO-028

MYO-028 reduced viscosity formulations can be formulated using 1 mg/mlto 300 mg/ml of the MYO-028 antibody. The MYO-028 formulation generallyincludes between about 1 mM and about 50 mM calcium chloride ormagnesium chloride. The formulation can include 10 between about 5 mM toabout 25 mM histidine. The formulation can include between about 1% toabout 5% (w/v) sucrose or trehalose. The pH of a MYO-028 formulation isgenerally between about 5.5 and about 6.5. In one specific example, aMYO-028 antibody formulation comprises 50 hs mg/ml of the antibody, 10mM histidine, 5% sucrose, and has a pH of 6.5. In another specificexample, a MYO-028 antibody formulation comprises 50 mg/ml of theantibody, 10 mM calcium chloride or magnesium chloride, 10 mM histidine,5% sucrose, and has a pH of 6.5.

J695

J695 reduced viscosity formulations can be formulated using 1 mg/ml to300 mg/ml of the J695 antibody. A J695 formulation generally includesbetween about 1 mM and about 50 mM calcium chloride or magnesiumchloride. The formulation can include about 5 mM to about 25 mMhistidine. The formulation may include about 1% to about 5% (w/v)sucrose or trehalose. In some instances, the formulation can includeabout 10 mM to about 25 mM methionine. In certain J695 formulations,between about 1% to about 5% (w/v) mannitol is added. The pH of theformulation is generally between 5.5 and 6.5. In a specific example, aJ695 antibody formulation comprises 100 mg/ml of the J695 antibody, 10mM histidine, 10 mM methionine, 4% mannitol, 1% sucrose, and has a pH of6.0. In another specific example, a J695 antibody formulation comprises100 mg/ml of the J695 antibody, 10 mM histidine, 10 mM methionine, 5mMcalcium chloride or magnesium chloride, 4% mannitol, 1% sucrose, and hasa pH of 6.0. In another specific embodiment, the J695 antibodyformulation comprises 100 mg/ml of the J695 antibody, 10 mM histidine,10 mM methionine, 10 mM calcium chloride or magnesium chloride, 4%mannitol, 1% sucrose, and has a pH of 6.0.

IMA-638

IMA-638 protein formulations can be formulated using 1 mg/ml to 300mg/ml of the IMA-638 antibody. A reduced viscosity formulationcontaining IMA-638 generally includes between about 1 mM and about 50 mMcalcium chloride or magnesium chloride. The formulation can includeabout 5 mM to about 25 mM histidine. The formulation can also includeabout 1% to about 10% (w/v) sucrose or trehalose. The pH of theformulation is generally between 5.5 and 6.5. In a specific example, theIMA-638 antibody formulation comprises 50 mg/ml of the IMA-638 antibody,10 mM histidine, 5% sucrose, and has a pH of 6.0. In another specificexample, the IMA-638 antibody formulation comprises 100 mg/ml of theIMA-638 antibody, 20 mM histidine, 10% sucrose, and has a pH of 6.0. Inanother specific example, the IMA-638 antibody formulation comprises 50mg/ml of the IMA-638 antibody, 5 mM calcium chloride or magnesiumchloride, 10 mM histidine, 10% sucrose, and has a pH of 6.0. In yetanother specific example, the IMA-638 antibody formulation comprises 100mg/ml of the IMA-63 8 antibody, 10 mM calcium chloride or magnesiumchloride, 20 mM histidine, 10% sucrose, and has a pH of 6.0.

Storage Methods

A reduced viscosity protein formulation described herein may be storedby any suitable method known to one of skill in the art. Non-limitingexamples of methods for preparing a reduced viscosity formulation forstorage include freezing, lyophilizing, and spray drying the proteinformulation.

In some cases, a reduced viscosity formulation is frozen for storage.Accordingly, it is desirable that the formulation be relatively stableunder such conditions, including when subjected to freeze-thaw cycles.One method of determining the suitability of a formulation for frozenstorage is to subject a sample formulation to at least two, e.g., threeto ten cycles of freezing (at, for example −20° C. or −80° C.) andthawing (for example by fast thaw at room temperature or slow thaw onice), determining the amount of LMW species and/or HMW species thataccumulate after the freeze-thaw cycles and comparing it to the amountof LMW species or HMW species present in the sample prior to thefreeze-thaw procedure. An increase in the LMW species or HMW speciesindicates decreased stability of a protein stored as part of theformulation. Size exclusion high performance liquid chromatography(SEC-HPLC) can be used to determine the presence of LMW and HMW species.A suitable formulation may accumulate undesirable HMW species or LMWspecies, but not to the extent that the presence of the HMW species orLMW species make the formulation unsuitable for its intended use.

In some cases, a formulation is stored as a liquid. Accordingly, it isdesirable that the liquid formulation be relatively stable under suchconditions, including at various temperatures. One method of determiningthe suitability of a formulation for liquid storage is to store thesample formulation at several temperatures (such as 2-8° C., 15° C., 20°C., 25° C., 30° C., 35° C., 40° C., and 50° C.) and monitoring theamount (e.g., change in percentage) of HMW species and/or LMW speciesthat accumulate over time. Additionally, the charge profile of theprotein may be monitored by cation exchange-high performance liquidchromatography (CEX-HPLC).

In general, the percentage of high molecular weight species or lowmolecular weight species is determined either as a percentage of thetotal protein content in a formulation or as a change in the percentageincrease over time (i.e., during storage), as is appropriate for theassay and parameter being determined. In general, and in non-limitingexamples, the change in the percentage of protein in high molecularweight species or low molecular weight species in a reduced viscosityformulation is not greater than 10%, e.g., not greater than about 8%,not greater than about 5%, or not greater than about 3% with respect tothe assayed parameter (e.g., time, temperature, additional compounds inthe formulation, lyophilization, or shaking).

Alternatively, a formulation can be stored after lyophilization. Theterm “lyophilization” as used herein, refers to a process by which thematerial to be dried is first frozen followed by removal of the ice orfrozen solvent by sublimation in a vacuum environment. An excipient(e.g., lyoprotectant) may be included in formulations that are to belyophilized so as to enhance stability of the lyophilized product uponstorage. The term “reconstituted formulation” as used herein, refers toa formulation that has been prepared by dissolving a lyophilized proteinformulation in a diluent such that the protein is dispersed in thediluent. The term “diluent” as used herein, is a substance that ispharmaceutically acceptable (safe and non-toxic for administration to ahuman) and is useful for the preparation of a liquid formulation, suchas a formulation reconstituted after lyophilization. Non-limitingexamples of diluents include sterile water, bacteriostatic water forinjection (BWFI), a pH buffered solution (e.g., phosphate-bufferedsaline), sterile saline solution, Ringer's solution, dextrose solution,or aqueous solutions of salts and/or buffers.

Testing a reduced viscosity formulation for the stability of the proteincomponent of the formulation after lyophilization is useful fordetermining the suitability of a formulation. The method is similar tothat described above for freezing, except that the sample formulation islyophilized instead of frozen, reconstituted using a diluent and thereconstituted formulation is tested for the presence of LMW speciesand/or HMW species. An increase in LMW species or HMW species in thelyophilized sample compared to a corresponding sample formulation thatwas not lyophilized indicates decreased stability in the lyophilizedsample.

In some cases, a formulation is spray-dried and then stored. For spraydrying, a liquid formulation is aerosolized in the presence of a dry gasstream. Water is removed from the formulation droplets into the gasstream, resulting in dried particles of the drug formulation. Excipientsmay be included in the formulation to (1) protect the protein during thespray-drying dehydration, (2) protect the protein during storage afterspray drying, and/or (3) give the solution properties suitable foraerosolization. The method is similar to that described above forfreezing, except that the sample formulation is spray-dried instead offrozen, reconstituted in a diluent and the reconstituted formulation istested for the presence of LMW species and/or HMW species. An increasein LMW or HMW species in the spray-dried sample compared to acorresponding sample formulation that was not lyophilized indicatesdecreased stability in the spray-dried sample.

Methods of Treatment

The reduced viscosity formulations described herein are useful aspharmaceutical compositions in the treatment and/or prevention of adisease or disorder in a patient in need thereof. The term “treatment”refers to both therapeutic treatment and prophylactic or preventativetreatment. Treatment includes the application or administration of thereduced viscosity formulation to the body, an isolated tissue, or cellfrom a patient who has a disorder, a symptom of a disorder, is at riskfor a disorder, or a predisposition toward a disorder, with the purposeto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the disorder, the symptom of the disorder, or thepredisposition toward the disorder. Those “in need of treatment” includethose who already have a disorder, as well as those in whom a disorderis to be prevented. The term “disorder” is any condition that wouldbenefit from treatment with a protein formulation described herein. Thisincludes chronic and acute disorders or diseases including thosepathological conditions that predispose the subject (patient) to thedisorder in question. Non-limiting examples of disorders to be treatedusing a formulation described herein include autoimmune disorders,inflammatory disorders, muscle wasting disorders, allergies, cancers,muscular dystrophy, sarcopenia, cachexia, Type II diabetes, rheumatoidarthritis, Crohn's disease, psoriasis, psoriatic arthritis, asthma,dermatitis, allergic rhinitis, chronic obstructive pulmonary disease,eosinophilia, fibrosis, and excess mucus production.

In one embodiment, the reduced viscosity formulation suitable for use asa pharmaceutical composition comprises an anti-myostatin antibody and aviscosity reducing agent. In one embodiment, the anti-myostatin antibodyis MYO-029. In other embodiments, the anti-myostatin antibody is MYO-022or MYO-028. The anti-myostatin antibody is generally at a concentrationof between about 0.5 mg/ml and about 300 mg/ml in the formulation. Inanother embodiment, the viscosity reducing agent is at a finalconcentration of between about 0.5 mM and 20 mM in the pharmaceuticalcomposition. In another embodiment, the viscosity reducing agent is at afinal concentration of between about 0.5 mM and 14 mM in thepharmaceutical composition. In another embodiment, the pharmaceuticalcomposition comprises an anti-myostatin antibody, a viscosity reducingagent, and a buffer wherein the pH of the formulation is between about5.5 to about 6.5. The pharmaceutical compositions described herein mayalso contain other proteins, drugs, and/or excipients. In particular,other proteins or substances useful for treating the disorder inquestion may be added to the formulation. Anti-myostatinantibody-containing pharmaceutical compositions are useful in thetreatment or prevention of disorders such as, but not limited to, musclewasting disorders, muscular dystrophy, sarcopenia, cachexia, and Type IIdiabetes.

In another embodiment, a pharmaceutical composition comprises ananti-IL-12 antibody and a viscosity reducing agent. In one embodiment,the anti-IL-12 antibody is J695. The anti-IL-12 antibody is generally ata concentration of between about 0.5 mg/ml and about 300 mg/ml in theformulation. In another embodiment, the viscosity reducing agent is at afinal concentration of between about 0.5 mM and 20 mM in thepharmaceutical composition. In another embodiment, the viscosityreducing agent is at a final concentration of between about 0.5 mM and14 mM in the pharmaceutical composition. In another embodiment, thepharmaceutical composition comprises an anti-IL-12 antibody, a viscosityreducing agent, and a buffer, wherein the pH of the formulation isbetween about 5.5 to about 6.5. The pharmaceutical compositionsdescribed herein may also contain other proteins, drugs, and/orexcipients. In particular, other proteins or substances useful fortreating the disorder in question may be added to the formulation.Anti-IL-12 antibody containing pharmaceutical compositions are useful inthe treatment or prevention of disorders such as, but not limited to,autoimmune disorders, inflammatory disorders, rheumatoid arthritis,Crohn's disease, psoriasis, and psoriatic arthritis.

In another embodiment, a pharmaceutical composition comprises ananti-IL-13 antibody and a viscosity reducing agent. In one embodiment,the anti-IL-13 antibody is IMA-638. The anti-IL-13 antibody is generallyat a concentration of between about 0.5 mg/ml and about 300 mg/ml in theformulation. In another embodiment, the viscosity reducing agent is at afinal concentration of between about 0.5 mM and 20 mM in thepharmaceutical composition. In another embodiment, the viscosityreducing agent is at a final concentration of between about 0.5 mM and14 mM in the pharmaceutical composition. In another embodiment, thepharmaceutical composition comprises an anti-IL-13 antibody, a viscosityreducing agent, and a buffer wherein the pH of the formulation isbetween about 5.5 to about 6.5. The pharmaceutical compositionsdescribed herein may also contain other proteins, drugs, and/orexcipients. In particular, other proteins or substances useful fortreating the disorder in question may be added to the formulation.Anti-IL-13 antibody containing pharmaceutical compositions are useful inthe treatment or prevention of disorders such as, but not limited to,asthmatic disorders, atopic disorders, chronic obstructive pulmonarydisease, conditions involving airway inflammation, eosinophilia,fibrosis and excess mucus production, inflammatory conditions,autoimmune conditions, tumors or cancers, and viral infection.

Administration

A reduced viscosity formulation described herein can be administered toa subject in need of treatment using methods known in the art, such asby single or multiple bolus or infusion over a long period of time in asuitable manner, e.g., injection or infusion by subcutaneous,intravenous, intraperitoneal, intramuscular, intraarterial,intralesional or intraarticular routes, topical administration,inhalation, or by sustained release or extended-release means. If theformulation has been lyophilized, the lyophilized material is firstreconstituted in an appropriate liquid prior to administration. Thelyophilized material can be reconstituted in, e.g., BWFI, phosphatebuffered saline, or the same formulation the protein had been in priorto lyophilization.

Parenteral compositions can be prepared in dosage unit form for ease ofadministration and uniformity of dosage. “Dosage unit form” as usedherein, refers to physically discrete units suited as unitary dosagesfor the subject to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the selected pharmaceuticalcarrier.

In the case of an inhalation method, such as metered dose inhaler, thedevice is designed to deliver an appropriate amount of a formulation.For administration by inhalation, the compounds are delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas, such as carbon dioxide, ora nebulizer.

Alternatively, an inhaled dosage form may be provided as a dry powderusing a dry powder inhaler.

A reduced viscosity formulation can also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 20th edition (supra).

Sustained-release preparations of the protein formulations describedherein can also be prepared. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the protein formulation. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers,and poly-D-(-)-3-hydroxybutyric acid. The sustained-release formulationsof the proteins described herein can be developed using e.g.,polylactic-coglycolic acid (PLGA) polymer due to its biocompatibilityand wide range of biodegradable properties. The degradation products ofPLGA, lactic and glycolic acids, can be cleared quickly within the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition.Liposomal compositions can also be used to formulate the proteins orantibodies disclosed herein.

Dosing

Toxicity and therapeutic efficacy of a formulation can be determined bypharmaceutical procedures known in the art using, for example, cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD₅₀/ED₅₀.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch formulations generally lies within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any formulationused in the method of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The appropriate dosage of the protein of the formulation will depend onthe type of disorder to be treated, the severity and course of thedisorder, whether the agent is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical, historyand response to the agent, and the discretion of the attendingphysician. A formulation is generally delivered such that the dosage isbetween about 0.1 mg protein/kg of body weight to 100 mg protein/kg ofbody weight. The formulation is administered to the patient at one timeor over a series of treatments. In one embodiment, a myostatin antibody(e.g., MYO-22, MYO-28, MYO-029) formulation is delivered to a patient inneed thereof at a dosage of 1 mg/kg to 10 mg/kg of body weight. Inanother embodiment, an IL-12 antibody formulation is administered to apatient in need thereof at a dosage of 1 mg/kg to 5 mg/kg of bodyweight. In a further embodiment, an IL-13 antibody formulation isadministered to a patient in need thereof at a dosage of about 0.5 mg/kgto about 5 mg/kg of body weight of the patient.

A formulation to be used for in vivo administration must be sterile. Aformulation can be rendered sterile for example, by filtration throughsterile filtration membranes, prior to, or following, formulation of aliquid or lyophilization and reconstitution. The therapeuticcompositions disclosed herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bag,or vial having a stopper pierceable by a hypodermic injection needle.

Articles of Manufacture

In another embodiment, an article of manufacture is provided thatcontains a formulation described herein and typically providesinstructions for its use. The article of manufacture comprises acontainer suitable for containing the formulation. Suitable containersinclude, without limitation, bottles, vials (e.g., dual chamber vials),syringes (e.g., single or dual chamber syringes), test tubes,nebulizers, inhalers (e.g., metered dose inhalers or dry powderinhalers), or depots. The container can be formed from a variety ofmaterials, such as glass, metal or plastic (e.g., polycarbonate,polystyrene, polypropylene). The container holds the formulation and thelabel on, or associated with, the container can indicate directions forreconstitution and/or use. The label may further indicate that theformulation is useful or intended for subcutaneous administration. Thecontainer holding the formulation may be a multi-use vial, which allowsfor repeat administrations (e.g., from 2-6 doses) of the formulation.The article of manufacture may further comprise a second containercomprising a suitable diluent (e.g., WFI, 0.9% NaCl, BWFI, or phosphatebuffered saline). When the article of manufacture comprises alyophilized version of a protein formulation, mixing of a diluent withthe lyophilized formulation will provide a final protein concentrationin the reconstituted formulation of generally at least 20 mg/ml. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

EXAMPLES Example 1 Viscosity of Antibody Formulations

Anti-β-amyloid peptide (anti-AB), anti-IL-13, anti-IL-12 (J695) andanti-myostatin (MYO-029) antibodies were formulated as described inTable 1. The viscosity of these antibody formulations was measured usingan Anton Paar Physica MCR301 cone and plate rheometer. Specifically, aCP25-1 (24.971 mm diameter, 1.002° angle) cone was used for all of themeasurements; the shear rate was constant at 898 1/s for a duration of100 seconds. Measurements were made every 10 seconds. The viscositymeasurements were performed at both 4° C. and 25° C. using a built-inPeltier temperature control unit. The liquid sample load on the platewas 90 μl. Each sample was analyzed in triplicate.

Table 1 below lists the viscosities of different antibodies at differentconcentrations and in different formulations. TABLE 1 Viscosity Conc. at25° C. Viscosity Antibody Formulation (mg/ml) (cP) at 4° C. (cP) Anti-AB10 mM Histidine, 10 mM Methionine, pH 6.0 160 5.18 9.77 Anti-IL-13 20 mMHistidine, 10% Sucrose, pH 6.0 100 7.18 16.44 J695 10 mM Histidine, 10mM Methionine, 100 6.81 18.43 4% Mannitol, 1% Sucrose, pH 6.0 MYO-029 20mM Histidine, 4% Sucrose, pH 6.0 70 2 5 MYO-029 10 mM Histidine, 10 mMMethionine, 114 14.71 61.74 2% Sucrose, pH 6.0 MYO-029 20 mM Histidine,20 mM Methionine, 127 50.95 Not Done 4% Sucrose, 0.2% Polysorbate-80, pH6.0 MYO-029 20 mM Histidine, 20 mM Methionine, 167 71.52 Not Done 4%Sucrose, 0.2% Polysorbate-80, pH 6.0

The data shown in Table 1 demonstrate that the viscosity ofanti-myostatin (MYO-029) is significantly higher compared to the otherantibodies listed in the Table. The viscosities of all of the antibodiesincreased at 4° C. This increase is proportionally much higher forMYO-029.

Example 2 Effect of Various Salts on the Viscosity of an MYO-029Antibody Formulation

MYO-029 antibody, at a concentration of 73 mg/ml, was formulated in 10mM histidine, 2% sucrose, pH 6.0. Concentrated solutions of salts (e.g.,calcium chloride, magnesium chloride, sodium chloride, and sodiumbiphosphate) were diluted into the MYO-029 antibody formulation using apipette. The effect of these salts on the viscosity of MYO-029 antibodyformulation was measured as described in Example 1. These data are shownin FIG. 1.

Both MgCl₂ and CaCl₂ at concentrations ranging from about 5 mM to about20 mM significantly reduced the viscosity of the MYO-029 antibodyformulation. NaCl and NaH₂PO₄, on the other hand, had little effect inthis range.

Thus, calcium chloride and magnesium chloride, at concentrations ofabout 5 mM to about 20 mM, are effective viscosity reducing agents forMYO-029 antibody formulations, unlike sodium chloride and sodiumbiphosphate.

Example 3 Effect of Calcium Chloride on the Viscosity of a J695 AntibodyFormulation

The viscosity of a J695 antibody formulation is measured at twodifferent J695 antibody concentrations, i.e., 100 mg/ml and 300 mg/ml.

The viscosity of the J695 antibody formulation at the higherconcentration will be higher than the viscosity of the J695 antibodyformulation at the lower concentration.

Calcium chloride is added to a final concentration of about 5 mM to 20mM to the 300 mg/ml J695 antibody formulation. In this case theviscosity of the antibody formulation is expected to decrease comparedto the J695 formulation without calcium chloride.

Accordingly, calcium chloride, at concentrations of about 5 mM to about20 mM, is effective as a viscosity reducing agent for J695 antibodyformulations.

Example 4 Effect of Calcium Chloride on the Viscosity of a MYO-028Antibody Formulation

MYO-028, another anti-myostatin antibody, was concentrated usingCentricon Ultrafree®-4 to a concentration of 95 mg/mL. Calcium chloridewas added to MYO-028 according to Table 2 below: TABLE 2 CaCl₂ Conc.MYO-028 (μL) CaCl₂ Solution (μL) Buffer (μL)  0 mM 316.9 0 8.125 25 mM316.9 4.06 4.06 50 mM 316.9 8.125 0

MYO-028 was formulated at 95 mg/mL in 10 mM histidine, 5% sucrose, pH6.5. The CaCl₂ solution consisted of 1 OmM histidine, 2% sucrose, 2MCaCl₂ The buffer solution consisted of 10 mM histidine, 5% sucrose, pH6.5.

The viscosity of these MYO-028 antibody formulations was measured usingthe same rheometer method as described in Example 1 with the additionaluse of a solvent trap to prevent evaporation, a 100 μL liquid sampleload of MYO-028 on the plate, and the test was performed at roomtemperature.

The data from these experiments are shown in FIG. 2.

The addition of CaCl₂ decreased the viscosity of a MYO-028 antibodyformulation at 25 mM and 50 mM CaCl₂ compared to a MYO-028 formulationlacking CaCl₂. These data demonstrate the suitability of CaCl₂ for useas an agent to reduce viscosity of a protein formulation, e.g., toformulate a reduced viscosity antibody formulation.

Example 5 Effect of Calcium Chloride on the Viscosity of a IMA-638Antibody Formulation

To test the effect of calcium chloride on the viscosity of an IMA-638antibody formulation, different amounts of calcium chloride were addedwith a pipette to aliquots of the IL-13 antibody, IMA-63 8. The IMA-63 8antibody aliquots had a protein concentration of approximately 150mg/mL. FIG. 3 provides a graphical depiction of the effect of calciumchloride on the viscosity of IMA-638 protein formulations.

The viscosity of the IMA-638 did not show the same reduction inviscosity as observed for MYO-029. These data demonstrate a method ofidentifying a suitable viscosity reducing agent for use with a proteinformulation.

Example 6 Effect of Calcium Chloride on the Stability of MYO-029Antibody

Addition of a compound (i.e., a viscosity reducing agent, e.g., CaCl₂)to a protein formulation could potentially affect the molecules'stability towards freeze-thaw-induced stresses. This effect could eitherbe detrimental, beneficial, or have no effect on a proteins' stabilityduring freezing and thawing.

To evaluate the effect of an agent (i.e., CaCl₂) on thefreeze-thaw-induced degradation of MYO-029 antibody, the molecule wassubjected to 10 freeze thaw cycles at −80° C. and 37° C., in thepresence or absence of 5 mM CaCl₂. MYO-029 drug substance was formulatedinto 10 mM histidine, 2% sucrose, in the presence or absence of calciumchloride by ultrafiltration and diafiltration. The final proteinconcentration was approximately 75 mg/mL. Twenty microliter aliquotswere frozen at −80° C. and thawed at room temperature. This was repeatedfor 5 and 10 freeze-thaw cycles. Samples were diluted 25-fold withformulation buffer and analyzed by measuring absorbance at 280 nm forprotein concentration and SEC-HPLC for the percentage of high molecularweight products (%HMW).

The effect of freeze-thaw-induced degradation was assessed by (i)protein recovery (absorbance at 280 nm), and (ii) percentage of highmolecular weight (% HMW) formation as determined by size exclusion-highperformance liquid chromatography (SEC-HPLC). HMW formation is the mostcommon degradation pathway for this molecule. The results of thesestudies are shown in FIG. 4A and FIG. 4B.

Compared to the corresponding control sample without CaCl₂, addition of5 mM CaCl₂ to the formulation did not have any effect on proteinrecovery or % HMW formation. Thus, the addition of calcium chloride doesnot appear to impact the stability of MYO-029 antibody formulations.This indicates that suitability of CaCl₂ for use as a viscosity reducingagent in a protein formulation, e.g., in a reduced viscosity antibodyformulation.

Example 7 Effect of Calcium Chloride on the Stability of a MYO-029Antibody Formulation

Addition of CaCl₂ to a protein formulation could potentially affect themolecules' liquid stability over time. This effect could either bedetrimental, beneficial, or have no effect on the proteins' stabilityduring storage.

To evaluate the effect of this agent on the liquid stability of MYO-029on heat-induced degradation, formulations containing MYO-029 weresubjected to storage at 50° C. for up to seven days. Aliquots were takenat various time points and analyzed for protein concentration byabsorbance at 280 nm and % HMW was analyzed by SEC-HPLC. The data areshown in FIG. 5A and FIG. 5B.

Compared to the control sample, addition of CaCl₂ to the formulation hadno negative effect on the stability of the protein in the liquid statestored at 50° C. The percentage of HMW in the drug substance alsoappeared to be slightly less in the material containing CaCl₂. Thesedata further demonstrate the suitability of using CaCl₂ as a viscosityreducing agent. They also demonstrate a method of determining thesuitability of an agent that reduces viscosity of a protein formulation,e.g., with respect to whether the agent has an effect on stability ofprotein in the formulation.

Example 8 Effect of Calcium Chloride on the Stability of LyophilizedMYO-029

Addition of an agent such as CaCl₂ to a protein formulation couldpotentially affect the proteins' lyophilized dosage forms stability overtime. This effect could be either detrimental, beneficial or have noeffect on the proteins' stability during storage.

To evaluate the effect of this excipient on the stability of lyophilizedMYO-029, a formulation containing the molecule is lyophilized both withand without (control) 5 mM CaCl₂ and is subject to storage at 50° C. and4° C. for four weeks. Vials are pulled weekly and analyzed for proteinconcentration by absorbance at 280 nm, percentage of HMW by SEC-HPLC,and charge distribution by cation exchange-high performance liquidchromatography (CEX-HPLC). Vial withdrawal volume and viscosity (onetime-point only) are also measured.

A. Viscosity of Reconstituted Drug Product

The viscosity of the MYO-029 drug product (which is measured insubstantially the same manner as in Example 1) at approximately 150mg/mL is reduced when 5 mM calcium chloride is present in theformulation.

B. Withdrawal Volume From the Vial

The amount of drug product that can be removed from the vial with a 1 mLsyringe and 21 G needle is improved when CaCl₂ is present in theformulation.

C. Protein Concentration

Compared to the control, addition of CaCl₂ to the formulation does notaffect protein recovery.

D. High Molecular Weight

Five mM CaCl₂ will not have any significant effect on the percentage ofHMW species that is formed after four weeks of storage at 4° C. However,at 50° C., the rate of HMW formation is expected to be significantlyreduced compared to the control.

E. Charge Distribution

The stability time-points are analyzed by CEX-HPLC, a chromatographictool used to study charge differences in proteins. In CEX-HPLC, the morenegatively charged molecules elute earlier than the more positivelycharged molecules. This method is used to detect deamidation ofasparagines residues to either aspartic or iso-aspartic acid.Deamidation results in an increase in the proteins' net negative charge,and it will elute earlier from the HPLC column. In this experiment, theeffect of calcium chloride has on protein degradation resulting in acharge change different from that of the control is investigated.Compared to the control without calcium chloride, the same chargechanges are expected to occur over time. Thus, CaCl2 is expected to haveno effect on the charge distribution of MYO-029 at both storagetemperatures.

In summary, compared to a control sample, addition of CaCl₂ to aformulation will have no significant negative effect on the stability ofthe protein in the formulation relative to the no calcium chlorideexperimental control in the lyophilized state when stored at 4° C. and50° C. In some instances, CaCl₂ is found to be beneficial to thestability of the protein.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for reducing the viscosity of a protein formulation, themethod comprising; (a) providing a protein formulation; and (b) addingcalcium chloride or magnesium chloride to the protein formulation to aconcentration of between about 0.5 mM and about 20 mM, wherein theviscosity of the protein formulation with the calcium chloride ormagnesium chloride is reduced compared to the viscosity of a proteinformulation without the viscosity reducing agent.
 2. The method of claim1, wherein the protein is selected from the group consisting of anantibody, an Ig fusion protein, a receptor, a transcription factor, anenzyme, a ligand, and a biologically active fragment thereof.
 3. Themethod of claim 1, wherein the protein is an antibody or a biologicallyactive fragment thereof.
 4. The method of claim 1, wherein the proteinis an Ig fusion protein.
 5. The method of claim 1, wherein the proteinis an antibody and the antibody is an anti-myostatin antibody, ananti-IL-12 antibody, or an anti-IL-13 antibody.
 6. The method of claim5, wherein the anti-myostatin antibody is MYO-029, wherein theanti-IL-12 antibody is J695, and wherein the anti-IL-13 antibody isIMA-638.
 7. The method of claim 1, wherein the viscosity of the proteinformulation is reduced by at least about 5% compared to the viscosity ofthe formulation in the absence of the viscosity reducing agent.
 8. Areduced viscosity formulation, comprising: (i) a protein; (ii) aviscosity reducing agent at a concentration of between about 5 mM andabout 20 mM in the formulation, wherein the viscosity reducing agent isnot sodium chloride or sodium biphosphate; and (iii) a buffer; whereinthe pH of the formulation is about 5.5-6.5.
 9. The reduced viscosityformulation of claim 8, wherein the protein is selected from the groupconsisting of an antibody, an Ig fusion protein, a receptor, atranscription factor, an enzyme, a ligand, and biologically activefragments thereof.
 10. The method of claim 8, wherein the protein is anantibody or a biologically active fragment thereof.
 11. The method ofclaim 8, wherein the protein is an Ig fusion protein.
 12. The proteinformulation of claim 8, wherein the viscosity reducing agent is calciumchloride, or magnesium chloride.
 13. An anti-myostatin antibodyformulation, comprising: (i) an anti-myostatin antibody or a myostatinbinding fragment thereof; (ii) a viscosity reducing agent; and (iii) abuffer, wherein the pH of the formulation is about 5.5-6.5.
 14. Themethod of claim 13, wherein the anti-myostatin antibody is a monoclonalantibody.
 15. The method of claim 14, wherein the anti-myostatinantibody is a humanized monoclonal antibody.
 16. The method of claim 14,wherein the anti-myostatin antibody binds myostatin with a K_(d) ofabout 6×10⁻¹¹ M as determined by Biacore™.
 17. The method of claim 14,wherein the anti-myostatin antibody is selected from the groupconsisting of MYO-022, MYO-028, and MYO-029.
 18. The method of claim 13,wherein the anti-myostatin in the formulation is at a concentration ofabout 25 mg/ml to about 400 mg/ml.
 19. The method of claim 13, whereinthe viscosity reducing agent is calcium chloride or magnesium chloride.20. The method of claim 13, wherein the viscosity reducing agent iscalcium chloride at a concentration of about 5 mM to about 20 mM. 21.The method of claim 13, wherein the buffer is histidine buffer at aconcentration in the formulation of about 4 mM to about 60 mM.
 22. Themethod of claim 13, wherein the formulation further comprises acryoprotectant.
 23. The method of claim 22, wherein the cryoprotectantis sucrose or trehalose at a concentration in the formulation of about0.5% to about 5% (weight/volume).
 24. The method of claim 13, whereinthe formulation further comprises a surfactant at a concentration in theformulation of about 0% to 0.2% (weight/volume).
 25. The method of claim24, wherein the surfactant is polysorbate-20 or polysorbate-80.
 26. Themethod of claim 13, wherein the formulation further comprises ananti-oxidant.
 27. The method of claim 26, wherein the anti-oxidant ismethionine, and the concentration of methionine in the formulation isbetween about 2 mM and about 20 mM.
 28. The anti-myostatin antibodyformulation of claim 13, wherein (i) the anti-myostatin antibody is afully humanized anti-myostatin antibody at a concentration of about 20mg/ml to about 400 mg/ml; (ii) the viscosity reducing agent is calciumchloride or magnesium chloride at a concentration of about 5 mM to 20mM; and (iii) the buffer is a histidine buffer at a concentration ofabout 5 mM to about 20 mM, wherein the pH of the formulation is about6.0.
 29. The formulation of claim 13, wherein the formulation islyophilized.
 30. The formulation of claim 13, wherein the viscosity ofthe formulation is reduced by at least about 5% compared to aformulation lacking the viscosity reducing agent.
 31. A pharmaceuticalcomposition for the treatment of a disorder selected from the groupconsisting of muscular dystrophy, sarcopenia, cachexia, and Type IIdiabetes, wherein the pharmaceutical composition comprises ananti-myostatin antibody formulation of claim
 9. 32. A method of treatinga disorder selected from the group consisting of muscular dystrophy,sarcopenia, cachexia, and Type II diabetes, the method comprisingadministering a pharmaceutically effective amount of an antibodyformulation comprising: (i) an anti-myostatin antibody or a myostatinbinding fragment thereof; (ii) a viscosity reducing agent; and (iii) abuffer, wherein the pH of the formulation is about 5.5-6.5.
 33. Themethod of claim 32, wherein the antibody formulation is administered byinjection, intravenous infusion, or pulmonary administration via anebulizer or as a dry powder.
 34. A kit comprising a containercontaining the formulation of claim
 1. 35. The kit of claim 34, furthercomprising directions for administration of said formulation.
 36. Amethod for reducing the viscosity of a protein formulation, the methodcomprising (i) providing a protein forumlation; and (ii) adding aviscosity reducing agent to the protein formulation, wherein theviscosity reducing agent is calcium chloride or magnesium chloride, andwherein the concentration of the viscosity reducing agent in theformulation is between about 1 mM and about 25 mM.
 37. A method foridentifying a reduced viscosity protein formulation, the methodcomprising (i) providing a protein formulation; (ii) adding a viscosityreducing agent to the protein formulation, wherein the viscosityreducing agent is calcium chloride or magnesium chloride, and whereinthe concentration of the viscosity reducing agent in the formulation isbetween about 1 mM and about 25 mM, thereby forming a potential reducedviscosity formulation; and (iii) determining the viscosity of thepotential reduced viscosity protein formulation, wherein when theviscosity of the potential reduced viscosity protein formulation isreduced compared to the viscosity of the protein formulation without theviscosity reducing agent, the formulation is a reduced viscosityformulation.
 38. The method of claim 1, wherein the method furthercomprises determining the stability of the protein formulation.
 39. Themethod of claim 37, wherein the method further comprises determining thestability of the protein formulation.
 40. The method of claim 39,wherein the stability of the protein formulation is determined afterfreeze-thawing the protein formulation, after storage of the proteinformulation at 50° C. for 1-7 days, or after lyophilization.
 41. Themethod of claim 39, wherein stability is determined by assaying thepercentage of high molecular weight species, the percentage of lowmolecular weight species, or the charge distribution of the proteinformulation compared to a control.